DESC:COMPLETE SPECIFICATION
Title: KITs for easy and effective venom Detection
Technical field of Invention:
The present invention is an easy to operate qualitative detection kits and related process for the identification of Venomous and Non-venomous bites from the bite cases. The kit can also qualitatively detect the Species responsible for the bite, using the body fluids such as blood, serum, plasma, saliva, urine and bite site secretions present in the victim's body as described herein. The present invention replaces the conventional non-specific diagnostic and symptomatic strategy and thereby the delay of Anti-Venom injection to the envenomed victims can be avoided. Conventionally, the clinicians will have to wait for the non-specific diagnostic and symptomatic results for the envenomation confirmation. This can be avoided by this novel invention, by early detection and thereby enabling administration of quick and effective treatment to the envenomed victims. It can also replace the conventional non-specific anti-venom treatment strategy to a specific treatment modality. This will significantly reduce the allergen load on the patient and associated side effects due to non-specific diagnostic detection assays. Further, the present invention resolves longstanding problem and much needed solution with far reaching benefit which reaches out for the benefit of civilians.
Background and prior art:
Bite envenoming is a potentially life-threatening disease caused by toxins in the bite of various venomous species. Of the 2500-3000 species of snakes distributed world-wide, about 500 are venomous. Over and above this there are many number of other venomous species capable of envenoming humans as well as animals, which can cause life threats to the victims. Based on their morphological characteristics including arrangement of scales, dentition, osteology, myology, sensory organs etc., species are categorized into families. The major families of venomous snakes are Atractaspididae, Elapidae, Hydrophidae and Viperidae. The main families in the Indian subcontinent are: Elapidae which includes Spectacled cobra, King cobra, Common Krait, etc., Viperidae which includes Russell's viper, Pit viper, Saw-scaled viper, etc., and Hydrophidae (the sea snakes). Of the 52 venomous species in India, majority of bites and consequent mortality is attributable to "Big four" species viz. Naja naja (Spectacled cobra), Daboia russelii (Russell's viper), Bungarus caeruleus (Common Krait) and Echis carinatus (Saw-scaled viper). Other venomous species include, scorpions, spiders, centipedes, various kinds of lizards, etc. The bite envenomation remains a public health concern in many countries especially India, concerning humans as well as animals.
Populations in these regions experience high morbidity and mortality because of poor access to health services, which are often suboptimal, and, in some instances, a scarcity of antivenom, which is the only specific treatment. A large number of victims survive with permanent physical sequelae due to local tissue necrosis and, no doubt, psychological sequelae. Because most bite victims are young, the economic impact of their disability is considerable. Worldwide more than 5 million bites are reported per year, with 2% leading to severe sequelae. In animal bite cases, many a time the realisation comes very late and hence the mortality will be very high.
India had the largest number of reported venomous bites and deaths. India has the highest number of deaths due to venomous bites in the world, around 1.2 million snake bite deaths (representing an average of 58,000 per year) from 2000 to 2019 with nearly half of the victims aged 30-39 and over a quarter being children under 15. The states with largest number of snake bite cases include Bihar, Jharkhand, Madhya Pradesh, Odisha, Uttar Pradesh, Rajasthan, Gujarat and Telangana. In 2017, snake bite was recognized by World Health Organization as a neglected tropical disease. Snake venom is the most complex of all poisons, is a mixture of enzymatic and non-enzymatic compounds as well as other non-toxic proteins including carbohydrates and metals.
Antivenom (or antivenin or antivenene) is a biological product used in the treatment of venomous bites or stings. Antivenom is created by milking venom from the desired snake, spider or insect. The venom is then detoxified and injected into a horse, sheep, goat or cat. The subject animal will undergo an immune response to the venom, producing antibodies against the venom's active molecule which can then be harvested from the animal's blood and used to treat envenomation. Antivenom can be classified into monovalent (when they are effective against a given species' venom) or polyvalent (when they are effective against a range of species, or several different species at the same time). Conventional clinical practice is to administer polyvalent anti- venom (anti venom), usually of horse origin at the time of hospitalization of the victim against Big Four venomous snake species. This often causes severe anaphylaxis reaction in the victim, (seen in up to 30% of the recipients worldwide) demanding secondary treatment. Acute pulmonary edemas, cerebellar ataxia and uveitis (an immunological complication) are some of the complications following polyvalent anti- venom (anti venom).
One of the major problems of the envenomation is the lack of specific species venom detection platforms. Currently, we don’t have any qualitative and quantitative detection diagnostics, we are still depending on symptomatic strategy and non-specific biochemical tests. It may cause delay in giving polyvalent anti venom or non-specific monovalent antivenom to victims. Because of non-specific diagnostics, we are still following the polyvalent treatment strategy to treat the bite patients.
The present invention, Qualitative platforms (Kits) which is mainly responsible for the detection of venomous and non-venomous bite identification and also qualitative detection of species responsible for the bite. The applied innovation includes two main platform for detection, one is modified Lateral flow Immuno Assay and another is Enzyme Linked Immuno Assay. Thus, the present invention proposes rapid identification platforms/kits for distinguishing venomous and non-venomous bites. In addition, this kits can be used by a layperson and is not limited to medical practitioner. This kits has potential to go up to species-specific and family based differentiation. In the present invention, rapid identification gives the differentiation output in less than 5 minutes, thus helping in early treatment of patients exposed to bite. Accordingly, the present invention proposes one-step rapid -envenomation detection kit, which can provide solution to the above listed limitations.
Though monovalent anti venoms are available, lack of a proper diagnostic system preludes this opportunity. Many attempts have been made worldwide to develop species specific diagnostic kit based on antibodies, but have not found successful mainly because of inter-species cross reactivity to the crude venom as also the lack of sensitivity related to the small quantities in which venom is injected.
FIGURES EXPLAINED
Fig:1 : Venom Milking Cartridge Design
1. Conical Top with Membrane attachment
2. Plastic vial – Plastic used as it has got non-sticky nature
3. Innovative membrane- a membrane which provides a medium to make the species feel it is biting a prey or enemy. The unique elasticity, tensile strength and thickness of the membrane become a stimulant to produce more venom from the biting species. The membrane specification varies from species to species.
Fig:2 : Bandage
1. Outer layer – A protective layer which enables the inner layer to stick to the wound and make it to be removed without any fuss.

2. Fluid absorbent layer- Highly hygroscopic material enabling fluid absorption to the maximum.
Fig: 3: Sample Diluent Cartridge
1. Filtration Pad
2. Filter attached Detachable cap were in the filtrate can be stored
3. Pressure applying module
4. Nozzle
5. The top portion where sample and buffer solution is inserted
Kit 1 - Lateral flow Immuno assay

Fig: 4: Immuno Chromatographic Strip
1. Adhesive card – Lowest portion of the strip which forms the base for the Immuno Chromatographic Strip.
2. Sample Pad – The portion where the sample is added for testing
3. Stacking Pad 1 – The material which controls the flow of the sample in a desired even mode and direction.
4. Conjugate Pad – The pad on which Conjugated antibody is placed for sample detection.
5. Stacking Pad 2- The material which controls the flow of the sample in a desired even mode and direction.
6. Cellulose Membrane- The material used to make the staking and conjugate pad enabling the capillary movement of filtrate / Sample. Cellulose membranes are used in lateral-flow assays as the substrate upon which immunocomplexes are formed and visualized to indicate the presence or absence of an analyte in a liquid sample.
7. Absorbent Pad – The excess sample is absorbed at the absorbent pad to stop overflow.
Fig: 5: Single cassette strip
Fig 5.a Single cassette strip with single lane– to detect presence of venom
1. - venom detection strip
2. -Sample well
Fig 5.b Single cassette strip with multi lane - to detect presence of venom and its corresponding families and species.
1. - venom detection strip
2. -Sample well
3. - Family detection strip
4. - Species detection strip
Fig: 6: Venom Lane
1. Adhesive card- Lowest portion of the strip which forms the base for the Immuno Chromatographic Strip
2. Sample Pad- The portion where the sample is added for testing
3. Stacking Pad 1 – The material which controls the flow of the sample in a desired even mode and direction.
4. Conjugate Pad – The pad on which Conjugated antibody is placed for sample detection.
5. Stacking Pad 2- The material which controls the flow of the sample in a desired even mode and direction.
6. Nitrocellulose Membrane- The material used to make the staking and conjugate pad enabling the capillary movement of filtrate / Sample. Nitrocellulose membranes are used in lateral-flow assays as the substrate upon which immunocomplexes are formed and visualized to indicate the presence or absence of an analyte in a liquid sample.
7. Absorbent Pad – The excess sample is absorbed at the absorbent pad to stop overflow.
8. Antibody with Conjugated Tag- Antibody bind with an enzyme which is responsible for color detection
9. Test Line Antibodies - Anti body coated portion where the filtrate react with the antibody and exhibit the presence of venom by a color lane.
10. Control Line Antibodies – Unbound conjugate antibody will be captured at the controlled line by the secondary antibody to form a conjugated-secondary antibody complex formation.
11. Sample- The filtrate used as sample
Fig: 7: Family Lane
1. Adhesive card- Lowest portion of the strip which forms the base for the Immuno Chromatographic Strip
2. Sample Pad- The portion where the sample is added for testing
3. Stacking Pad 1 – The material which controls the flow of the sample in a desired even mode and direction.
4. Conjugate Pad – The pad on which Conjugated antibody is placed for sample detection.
5. Stacking Pad 2- The material which controls the flow of the sample in a desired even mode and direction.
6. Nitrocellulose Membrane- The material used to make the staking and conjugate pad enabling the capillary movement of filtrate / Sample. Nitrocellulose membranes are used in lateral-flow assays as the substrate upon which immunocomplexes are formed and visualized to indicate the presence or absence of an analyte in a liquid sample.
7. Absorbent Pad – The excess sample is absorbed at the absorbent pad to stop overflow.
8. Elapidae Line- The portion where the antibodies corresponding to the Elapidae family is coated and this portion shows colour change when the venom of Elapidae family reacts with the antibody
9. Viperidae Line- The portion where the antibodies corresponding to the Viperidae family is coated and this portion shows colour change when the venom of Viperidae family reacts with the antibody
10. Control Line- Unbound conjugate antibody will be captured at the controlled line by the secondary antibody to form a conjugated- secondary antibody complex formation.
11. Sample- The filtrate used as sample
12. Antibody with Conjugated Tag- Antibody bind with an enzyme which is responsible for color detection

Fig: 8: Species Lane
1. Adhesive card- Lowest portion of the strip which forms the base for the Immuno Chromatographic Strip
2. Sample Pad- The portion where the sample is added for testing
3. Stacking Pad 1 – The material which controls the flow of the sample in a desired even mode and direction.
4. Conjugate Pad – The pad on which Conjugated antibody is placed for sample detection.
5. Stacking Pad 2- The material which controls the flow of the sample in a desired even mode and direction.
6. Nitrocellulose Membrane- The material used to make the staking and conjugate pad enabling the capillary movement of filtrate / Sample. Nitrocellulose membranes are used in lateral-flow assays as the substrate upon which immunocomplexes are formed and visualized to indicate the presence or absence of an analyte in a liquid sample.
7. Absorbent Pad – The excess sample is absorbed at the absorbent pad to stop overflow.
8. Spectacled Cobra Line - The portion where the antibodies corresponding to the species cobra is coated and this portion shows color change when the venom of cobra reacts with the antibody.
9. Common Krait Line- The portion where the antibodies corresponding to the species Common krait is coated and this portion shows color change when the venom of Common krait reacts with the antibody.
10. Saw scaled Viper Line- The portion where the antibodies corresponding to the species Saw scaled Viper is coated and this portion shows color change when the venom of Saw scaled Viper reacts with the antibody.
11. Russell’s Viper Line- The portion where the antibodies corresponding to the species Russell’s Viper is coated and this portion shows color change when the venom of Russell’s Viper reacts with the antibody.
12. Control Line- Unbound conjugate antibody will be captured at the controlled line by the secondary antibody to form a conjugated-secondary antibody complex formation.

Kit 2 - Enzyme Linked Immuno Assay
Fig: 9: Top view of innovative Enzyme Linked Immuno Assay Strip
1. Platform where wells are placed- These wells are coated with various antibodies and lyophilized conjugated specific antibodies. It facilitates more quantity of filtrate sample to be used for specific detections of venom, corresponding family or species.
2. Handle – Enabling proper hold and control on the strip

Fig 9(a): VENOM LANE – Immuno Strip
1. Blank- Well without any antibody or conjugate
2. Positive control- well coated with normal sheep IgG as capture antibody and rabbit anti sheep IgG (secondary antibody) or conjugate.
3. Negative Control- well coated with normal sheep IgG as capture antibody and anti- species antibody as conjugate
4. Venom Lane- Well with Anti body, raised from mixture of venom from different venemous species, coated and added corresponding lyophilized conjugated antibodies.

Fig 9(b): FAMILY LANE – Immuno Strip (Snake Family illustrated)
1. Family 1 (Elapidae) - The well coated with antibodies corresponding to the Elapidae family and added corresponding lyophilized conjugated antibodies. The residues in the well shows color change when the venom of Elapidae family reacts with the antibody
2. Family 2 (Viperidae) - The well coated with antibodies corresponding to the Viperidae family and added corresponding lyophilized conjugated antibodies. The residue in the well shows color change when the venom of Viperidae family reacts with the antibody
3. Negative Control – Well coated with normal sheep IgG as capture antibody and anti-species antibody as conjugate
4. Positive Control- Well coated with normal sheep IgG as capture antibody and rabbit anti sheep IgG (secondary antibody) as conjugate.
5. Blank - Well without any antibody or conjugate
Fig 9(c): SPECIES LANE– Immuno Strip (Snake species illustrated)
1. Common Krait (Bungarus caeruleus) – Well coated with the antibodies corresponding to the species Common krait and added corresponding lyophilized conjugated antibodies. The sample poured to this well reacts with the antibody and shows color change , if it contains the venom of Common krait.
2. Spectacled Cobra (Naja naja) - Well coated with the antibodies corresponding to the species Spectacled Cobra and added corresponding lyophilized conjugated antibodies. The sample poured to this well reacts with the antibody and shows color change, if it contains the venom of Spectacled Cobra.
3. Saw Scaled Viper (Echis carinatus)- Well coated with the antibodies corresponding to the species Saw Scaled Viper and added corresponding lyophilized conjugated antibodies. The sample poured to this well reacts with the antibody and shows color change, if it contains the venom of Saw Scaled Viper
4. Russell’s Viper (Daboia russelii ) -Well coated with the antibodies corresponding to the species Russell’s Viper and added corresponding lyophilized conjugated antibodies. The sample poured to this well reacts with the antibody and shows color change, if it contains the venom of Russell’s Viper.
5. Hump nosed pit viper (Hypnale hypnale) -Well coated with the antibodies corresponding to the species Hump nosed pit viper and the sample poured to this well reacts with the antibody and shows color change, if it contains the venom of Hump nosed pit viper.
6. Negative Control - Well coated with normal sheep IgG as capture antibody and anti-species antibody as conjugate
7. Positive Control - Well coated with normal sheep IgG as capture antibody and rabbit anti sheep IgG (secondary antibody) as conjugate
8. Blank - Well without any antibody or conjugate

DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to rapid detection kits for determining the presence of specific venom toxin in the victim’s body fluids/ samples which is essentially important for venomous and non-venomous differentiation and detection. And further, the venom toxins under the venomous families and species-specific detection can also be specifically incorporated into the kits for effective treatment strategy. This will reduce the error and also effective qualitative and quantitative methodology can lead to a specific treatment management strategy in venomous bite cases. The applied innovation kits are of two types:-
1. Novel lateral flow immunoassay - wherein the components are arranged in a defined manner to obtain the detection of venom in the sample to be detected accurately and mapped against its family and species.
2. Enzyme Linked Immuno Kit, - wherein detection wells are provided enabling more filtrate samples to be used for testing enabling even very low presence of venom in the sample to be detected accurately and mapped against its family and species.
The novel innovative kits and its unique process start with the novel method of collecting venom from various species. The specimens collected from the wild condition will be placed in the recognized parks or serpentariums, and acclimatized for 6 months.
After acclimatization, the specimens will be fasted for one week and venom will be milked from the fasted specimen with minimal stress. The venom milked in a specifically designed polypropylene container (Fig.1), fitted with a unique elastic membrane having a specific thickness and tensile strength which suit the species and age of species selected to milk, made from thick parafilm, mimicking the natural medium of their respective bites, like skin of prey. The so extracted venom is maintained in a specific low temperature (0oC – 20oC) container for protein stability. The milked venom will be then lyophilized and used for purification steps.
The crude venom of each species will be used for isolation and purification of specific protein separation. The specific proteins/antigens from each species and families will be isolated and purified using chromatographic techniques.
For the detection of venomous and non-venomous species, the major proteins of each species will be pooled together and used for antibody raising. Phospholipase A2, Metalloproteases, Serine proteases, L-amino acid oxidase, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor, mainly seen in Viperidae and Elapidae families in case of snake species, will be isolated and purified for antibody raising steps. Similar to this proteins and non-proteinaceous components from various species will be selected for antibody raising process as part of detection.
For Family based detection platform, Species-specific Phospholipase A2, Metalloprotease, Serine protease and L-amino acid oxidase proteins under the family Viperidae and species-specific Phospholipase A2, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor from the Elapidae family will be isolated and purified using controlled conditions. For Family specific detection, especially in the present invention, Viperidae and Elapidae, responsible for major deaths in Indian subcontinent, the platform will use family specific proteins in the case of Snakes. Further, the antigens from other families can also be used for detection. This can vary according to the presence of various species and threat of bite at various locations and countries. Also, other similar family detection process flow can also be applied for the detection of other venomous animals, like scorpions, insects, spiders, etc.
The species-specific proteins in each species will be isolated and purified for antibody raising steps. The isolated proteins/antigens will be used specifically against the detection platform development strategy. The species-specific proteins isolated will be used for species-specific detection.
The isolated and purified proteins (antigens) will be used for raising of the antibodies in sheep. Further, the primary antibodies can also be raised in horse, goat, shark, camel and avian models. The raised antibodies will be purified and specific non-cross reactive antibodies will be separated using family and species-specific venom column based isolation chromatographic models.
According to the present invention, the antibodies are species-specific primary antibody specific to venom. For example in the case of Snakes, for identifying Elapidae in the sample, antibodies that are raised against major Elapidae venomous species in animal models and called as anti-Elapidae antibodies as these snakes are known to have neurotoxins in their venom. Similarly, primary antibodies specific to Viperidae are developed as they are known to have hemotoxins in their venom. Thus, the primary antibodies are developed in animal models against the two major venomous families. Further, for raising these antibodies whole venom or different isolated antigenic proteins can be used.

According to the present invention, the secondary antibodies are preferably antibodies raised in specific and required animal models against the primary antibodies.
The patient’s samples will be collected in a novel way. An adhesive bandage (Fig.2) with an outer and inner layer. The inner layer containing a fluid absorbent layer and adhesive layer, which was protected by a covering layer, which will maintain the stability of the absorbent and adhesive layers. The bandage affixed on the bite site has to be pulled off. The bandage will have fluid sample absorbed in it. This is one of the model of retrieving the fluid samples from the bite site, which contain maximum amount of foreign protein samples, including the venom toxins, if it is a venomous bite. In addition to the bite site fluids, the blood, serum, plasma and urine will be collected by conventional methodology for further analysis using this innovative rapid detection kit.
The samples collected using various methods will be transferred into a novel container (Fig.3), where it will be then mixed with premixed components for one minute duration. It will be transferred to the sample addition site, with a pressure applying module. The debris and high molecular weight substances will be removed with the support of the filtration pad placed on the bottom portion of container.
Qualitative Assay Model – Kit 1: The figures used are just for illustration.
Lateral flow immunoassay:
According to the present invention, the lateral flow immunoassay is used for rapid detection of venom in victim’s sample and further to differentiate the venom toxins between various species, the families which are responsible for major envenomation causing bite deaths. In addition to that the assay is designed for species–specific detection under each families. As illustrated in Fig.4, the kit includes an immuno-chromatographic strip comprising of adhesive card (1), sample pad (2), stacking pad 1 and 2 (3, 5), a gold conjugate pad (4), nitrocellulose membrane (6), and absorbent pad (7). The materials make contact with each other in succession from sample pad, to stacking pad 1, to conjugate pad, to stacking pad 2, to nitrocellulose membrane, to absorbent pad. The stacking pad will be placed after sample pad is a novel design which is very essential for stacking the samples and filtration of the samples added for analysis. The conjugated molecule stacking will be done by the stacking pad placed after the conjugate pad. The sample is added to the sample pad, the materials comprising the strip act as wick and cause the sample to travel laterally across the strip until it reach the distal end, the absorbent pad (7). Conjugate and sample pads should be hydrophobic and importantly have good adsorption capacity. The nitrocellulose material consists of different detection lines referred as control line and test lines. In order to avoid the uneven transport of molecules especially the sample load, an innovative inclination is designed in the immune-chromatographic strip. Also further, a vacuum based sucking platform can be incorporated for the smooth and even transaction of molecules in the strip. 1. Venom lane; 2. Family Lane and 3. Species Lane.
Kits are developed based on the incorporation of the strips specifically designed. The venom lane based immune-chromatographic strip will be placed in a single cassette (Fig.5 (a)), which will be specifically for venom detection (Venomous or Non-venomous identification) from the samples. All three lanes can be placed in a single cassette (Fig.5 (b)), where each having separate detection models and antigen-antibody interaction. Further, each lane can also be incorporated in separate cassette as part of analysis and region wise strategy.
As illustrated in Fig. 6 of the present invention, the venom lane, the sample is passed from sample pad (2) to conjugate pad (4) having antigen specific antibody labelled with nanoparticle, where the sample venom antigen reacts with the conjugated primary antibodies and forms a conjugate antibody-antigen complex (hereinafter the conjugate complex). This conjugate complex along-with the unbound conjugate antibodies travels towards nitrocellulose membrane via capillary movement. As the conjugate complex travels further, the primary antibodies captures the conjugate complex. The conjugate complex on binding with primary antibodies contains antigen sandwiched between labelled antibody and primary antibody forming primary antibody-conjugate complex. At the same time, unbound conjugate antibody travels further and is captured at control line by secondary antibody to form secondary antibody conjugate formation. The color indication at control line indicates the travelling of sample across the membrane. The excessive sample and conjugate antibodies are further collected in absorbent pad.
The primary antibodies used in venom lane are anti- venom antibodies raised against major venom proteins from venomous species. Here, the major venom proteins of each species desired will be pooled together and used for antibody raising. In the case of Snakes, Phospholipase A2, Metalloproteases, Serine proteases, L-amino acid oxidase, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor, mainly seen in Viperidae and Elapidae families, will be isolated and purified for next antibody raising steps. The coloured line in the test line denotes presence of venom in the sample and, while control line should always appear regardless of the presence or absence of antigen in the sample. After passing these reaction lines, the sample enters the final porous material, the absorbent pad that simply acts as a waste container.
As illustrated in Fig. 7 of the present invention, the Family lane, the primary antibodies are raised from family specific venom proteins. Species-specific Phospholipase A2, Metalloprotease, Serine protease and L-amino acid oxidase proteins under the family Viperidae and species-specific, eg. Phospholipase A2, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor from the Elapidae family will be isolated and purified in the case of Snakes. For Family specific detection, especially in the present invention, Viperidae and Elapidae, responsible for major deaths in Indian subcontinent, the platform will use Snake family specific proteins. Similarly, the antigens from other families can also be used for detection. This can vary according to the presence of various species and threat of bite at various locations and countries. Also, other similar family detection process flow can also be applied for the detection of other venomous animals, like scorpions, insects, spiders, etc.
Also further in Species Lane, Fig.8, for primary antibodies, where the species-specific proteins in each species will be isolated and purified for primary antibody raising steps. The isolated proteins/antigens will be used specifically against the

detection platform development strategy. The species-specific proteins isolated will be used for species-specific detection. Different species can be identified with this lane, which is essentially important for effective treatment strategy.
According to one of the embodiments of the present invention, the lateral flow immunoassay works on an immune-chromatographic principle. In the present invention, a whole blood sample containing the venom toxin collected from the victim’s affected area is introduced on the sample pad. The sample travels laterally via capillary movement towards conjugate pad. The venom antigen (if pre
sent) will form an antigen antibody complex. The conjugate pad has immobilized primary antibodies coated with nanoparticles (i.e. specific antivenom antibodies raised against the specific venomous species) on the conjugate pad. This so formed antigen+conjugate-antibody complex, travels towards the cellulose membrane. The cellulose membrane has indicator lines referred as test lines and control line. The antigen has multiple binding sites, thus the primary antibodies on test lines are bound to antigen complexed with conjugate antibodies. This binding results in colorations. The binding forms a red coloration on the test line indicating the presence of venom antigen. The intensity of the red coloration on the test line is directly proportional to the amount of venom toxins present in the sample. An unbound conjugate antibody travels further on the membrane and is captured at control line by secondary antibody.
These secondary antibodies are produced specific to primary antibodies. In the control line, the conjugated antibodies forms complex with the secondary antibodies resulting in red coloration. The red line on the control line acts as a positive control to assure that functional, conjugated antibody has travelled throughout the system. A coloured line in the control line should always appear regardless of the presence or absence of venom antigen in the sample.
Qualitative Assay Model 2:
Enzyme Linked Immuno Assay Kit:
The Enzyme linked immuno assay kit is used for the in vitro detection and immunological identification (immunotyping) of venom in samples from bite sites, urine, plasma, blood or other tissue and body fluids in cases of bite victims, whether human or animal. . The primary purpose of this kit is to find out whether the bite sustained by a victim is venomous or non-venomous. This kit is designed in a way that it can also determine the family specific and species-specific identification of species responsible for the bite, which can assist in choosing the antivenom therapy to match the immunotype of venom involved in a clinically significant bite. The test gives a visual, qualitative result within 15 minutes and can detect as little as 0.01pg/mL of venom in a sample.
Immuno-chromatographic microtitre well strips made of polystyrene will be designed in three lane models, 1. Venom lane; 2. Family Lane and 3. Species Lane.
The Enzyme linked immune assay kit consists of a well holder( Fig 9.1) which can hold detachable wells as per the kit design and a handle to hold the whole kit (fig 9.2)
The major venom proteins of each species will be pooled together and used for antibody raising. For example in the case of snakes, Phospholipase A2, Metalloproteases, Serine proteases, L-amino acid oxidase, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor, mainly seen in Viperidae and Elapidae families, will be isolated and purified for next antibody raising steps, which will be used in venom lane immuno well strip development.
For Family specific detection, Species-specific Phospholipase A2, Metalloprotease, Serine protease and L-amino acid oxidase proteins under the family Viperidae and species-specific Phospholipase A2, Three Finger toxins, Acetylcholine Esterase and Cobra Venom Factor from the Elapidae family will be isolated and purified using controlled conditions in the case of Snakes. For Family specific detection, especially in the present invention, Viperidae and Elapidae, responsible for major deaths in Indian subcontinent, the platform will use family specific proteins if it is for snake venom detection. Further, the antigens from other families can also be used for detection in Family lane strip development. The species and families of the samples can vary as per the location and risk of bite from location to location or geography to geography. Same formation can be repeated using venom contents from various species and families capable of envenomation.
The species-specific proteins in each species will be isolated and purified for antibody raising steps. The isolated proteins/antigens will be used specifically against the detection platform development strategy. The species-specific proteins isolated will be used for species-specific detection in Species lane models.
The isolated and purified proteins (antigens) will be used for raising of the antibodies in sheep. Further, the primary antibodies can also be raised in horse, goat, shark, camel and avian models. The raised antibodies will be purified and specific non-cross reactive antibodies will be separated using family and species-specific venom column based isolation chromatographic models. The antibodies raised in each detection models will be used for microtiter well coating and conjugate preparation. For conjugate preparation, the antibodies raised will be labelled with a specific enzyme, horse radish peroxidase.
The kits comprised of three types of well microtiter test strips containing lyophilised conjugate, a cassette (a strip holder fig 9.1), a sample Diluent, chromogen solution and a substrate solution. The strips are venom lane strip, family lane strip and Species Lane strip, specifically for the identification of venom toxins under each category. The strips will be placed in an innovative cassette Fig.9. which is essentially important for avoiding cross contamination during identification steps.
The venom lane strip [Fig. 9 (a)] contain detachable wells, of which the first well is blank, without any antibody or conjugate; second well is positive control with normal sheep IgG as capture and rabbit anti-sheep IgG (secondary antibody) conjugate, third well is negative control with normal sheep IgG as capture and an anti-species conjugate, and the final well contain capture antibody and conjugate

to major venomous species like Naja naja (Spectacled Cobra), Daboia russelii (Russell’s viper), Echis carinatus (Saw scaled viper), Bungarus caeruleus (Common Krait) ,Hypnale hynale (Hump-nosed pit viper), if it is the case of snakes. Further, the more medically significant venomous species in region wise mode can also be used. Antibodies raised against these species will be incorporated in the final well as part of this design.
The family lane strip illustrated in Fig.9 (b), contain five detachable wells, of which the first well is blank; second well and third well is positive control and negative control respectively. Fourth well is designed for Viperidae family detection, where Viperidae specific proteins will be detected with the help of antibodies and conjugates raised against the family specific proteins. Fifth well will detect Elapidae family species contain antibodies and conjugates against the family specific proteins of the same in the case of Snakes. This type of family based detection can also be applicable to other venomous species other than snakes.
The Species-specific Lane strip illustrated in (Fig.9 (c) contain eight detachable wells; in consistent with the previous lanes, the first well, second and third well will be blank, positive control and negative control with the same content design and concentration. Fourth well will contain capture antibody and conjugate to Hypnale hypnale (Hump-nosed pit viper), and similarly the next four wells (from 5th to 8th) contain reagents to identify venoms from Daboia russelii (Russell’s viper), Echis carinatus (Saw scaled viper), Naja naja (Spectacled Cobra) and Bungarus caeruleus (Common Krait), in the case of snake species detection. This is applicable for the species level detection of other venomous animals like scorpions, spiders, centipedes, etc.
The sample collection from bite sites, urine, plasma, blood or other tissue and body fluids, will be done and placed in the innovative sample vial as previously discussed using Fig.2 and Fig.3 using a sample diluent. It will be added to each well followed by a 10 min incubation at room temperature. After that the wells will be washed at least five times under running tap water. After that one drop each of chromogen solution and peroxide solution will be added to each well and gently mixed. Wells will be observed for the development of a colour, which occurred within 10 min for positive test samples and the positive control. The negative control well will remain colourless.
,CLAIMS:
CLAIMS
We Claim:-
1. The innovative devices are kits for rapid detection of venom and for rapid identification of the family and species responsible for envenomation in victim’s body using body fluids/samples. Thus, a three layer of identification, detection of venom, identification of family and identification of species, which is responsible for envenomation is made possible in short span of time.
2. As claimed in claim 1: an innovative Immuno Chromatographic Strip (Fig 4) to identify the presence of venom in a bite site of the victim. It consists of an adhesive card (Fig 4.1), base of the strip; a sample pad (Fig 4.2), stacking pad 1 (Fig 4.3), to add the sample; conjugate pad (Fig 4.4) ,placed with conjugated antibody for sample detection; stacking pad 2 (Fig 4.5) to control the flow of the sample in a desired even mode and direction; Cellulose Membrane (Fig.4.6) enabling the complex visualization of antigen-antibody reaction and capillary movement of filtrate / Sample and absorbent pad (Fig.4.7), where excess sample is absorbed to restrict overflow. The antibody against pooled venom components from all desired venomous species are used for detection of venom in a bite site (Fig 6). The antibody against pooled family venom components is used for detection of family responsible for envenomation (Fig 7). The antibody against pooled species specific venom components is used for detection of specific species responsible for envenomation (Fig 8).

3. As Claimed in Claim 1: innovative Single cassette strips- one to detect the presence of venom (fig 5.a) and another to identify the presence of venom, differentiate the family as well as the species responsible for the envenomation (fig 5.b). The strips contain well/wells (Fig 5.2) for collection and distribution of sample. Strip/ strips (Fig 5.1) to detect the presence of venom, family (Fig 5 b.4) and species (Fig 5 b.3) coated with specific antibodies which is designed to react with the venom components in the sample to give line markers of detection (eg.T, V, E, RV, SS, CK, SC, etc. in the illustration) compared against the control (c), as per the strip used.
4. As claimed in claim 1: An unique Enzyme linked immuno assay kit for the in vitro detection and immunological identification (immunotyping) of venom in samples from bite sites, urine, plasma, blood or other tissue and body fluids in cases of bite victims, whether human or animal. The kit consists of a consists of a well holder ( Fig 9.1) which can hold detachable wells as per the kit design and a handle to hold the whole kit (Fig 9.2). The detachable wells can be coated with antibodies against, venom components pertaining to whole venom, family and species. The antibody against pooled venom components from all desired venomous species are used for detection of venom in a bite site (Fig 9 a). The antibody against pooled family venom components is used for detection of family responsible for envenomation (Fig 9 b). The antibody against pooled species-specific venom components is used for detection of specific species responsible for envenomation (Fig 9 c). The detachable wells facilitate usage of high quantity and multiple samples for testing, detecting venom, the family and species responsible for envenomation.

5. A unique device to collect the venom (Fig.1), made out of non-sticky material, which is having a conical shaped mouth with an extension of cylindrical tube fitted with a species specific lipid membrane at the mouth, which has specific thickness and tensile strength to suit the species and age of species selected to milk, mimicking the natural medium of their respective bites, like skin of prey. The unique container is designed to collect venom with minimal stress to the species and which gives maximum venom yield.
6. A unique process of pooling isolated major proteins and non-proteinaceous components, from identified family and species, as per topographical presence, to raise antibodies, for detection of venom. Similarly for raising family specific antibodies proteins and non-proteinaceous components isolated from various families are pooled. For raising species specific antibodies proteins and non-proteinaceous components isolated from various species are pooled.

7. A unique bandage (Fig 2) to collect venom from body fluid of the victim. It has an outer (Fig 2.1) and Fluid absorbent layer (Fig 2.2). The inside of the outer layer has an adhesive portion enabling the bandage to stick to the body. The fluid absorbent layer is to be attached to the bite site, which is protected by a covering layer, which will maintain the stability of the fluid absorbent layer. The fluid absorbent layer will absorb and keep the venom components and body fluids for being used as sample.
8. A unique Sample Diluent Cartridge (fig 3) with a top detachable portion (fig 3.5) where sample and buffer solution and the pressure applying module (fig 3.3) is inserted; a nozzle (fig 3.4), through which the filtered sample is extracted; a detachable filtration pad (fig3.1) is attached to the nozzle (fig 3.2) to where the buffered sample can be filtered, stored and extracted when as per usage.